1. Field of the Invention
The present invention relates to the release of bound biotin from a biotin-binding compound such as streptavidin (or avidin). In particular, the invention relates to a method of reversibly releasing a biotinylated moiety from a streptavidin (or avidin) coated support.
The strong interaction between streptavidin (or avidin) and biotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid) is well known. Indeed, the binding between streptavidin and biotin (dissociation constant, Kd approx. 10−15M) is regarded as one of the strongest known, non-covalent, biological interactions. The bond forms very rapidly and is considered to be stable under a wide range of pH, temperature and other denaturing conditions (Savage et al., Avidin-Biotin Chemistry: A Handbook, 1992:1-23, Rockford, Pierce Chemical Company). This has led to diverse and widespread applications using streptavidin-biotin or avidin-biotin technology.
2. Description of Related Art
Biotin-streptavidin is one of the most widely used affinity bindings in molecular, immunological and cellular assays. Streptavidin can be detected and quantitated with a high degree of sensitivity in such complexes, for example by labelling it with enzymes or with fluorescent, chemiluminescent or radioactive agents. Labelled streptavidin has, for example, been used to detect proteins on a cell surface, to visualize and quantitate blots, and to perform an enzyme-linked immunosorbent assay ELISA.
Streptavidin can also be readily immobilised at surfaces to capture biotinylated moieties, e.g. biotinylated molecules or cells. Such surfaces are used to detect and separate molecules or cells of interest from complex mixtures. The streptavidin-biotin interaction has thus found use in many of the separation, purification and isolation procedures known in the art, for example affinity chromatography, etc.
Immobilisation, particularly of oligonucleotides and nucleic acids, is frequently used in many molecular biological procedures and many of the commonly-used techniques, e.g. sequencing, in vitro amplifications, cDNA preparation, template preparation, etc., as well as nucleic acid purification, have been adapted for use on a solid phase, for example on a streptavidin-coated support. One particular area in which this is used is in the isolation of polymerase chain reaction (PCR) products, for example using streptavidin-immobilised magnetic microbeads (Hultman et al., Nucleic Acids Res. 17:4937-4946, 1989). This method is of great interest since it generally provides good yields and is easy to automate compared to traditional methods for purification of cycle sequencing products which often employ precipitation and/or centrifugation steps.
Most applications which use the biotin-streptavidin (or avidin) linkage rely on the essentially irreversible binding of biotin and streptavidin (or avidin). However, there are many cases in which release of bound biotin is desirable, e.g. to recover biotinylated molecules or cells.
A number of alternative strategies to disrupt or reverse the biotin-streptavidin linkage have previously been reported (Lee et al., Anal Biochem. 206:206-207, 1992, Elgar et al., DNA Sequence 2:219-226, 1992, and Conrad et al., Nucleic Acids Res. 20:6423-6424, 1992). However, these require harsh conditions, e.g. boiling in high salt conditions or use of formamide and EDTA heated to 94° C. for several minutes (Tong et al., Anal. Chem. 64:2672-2677, 1992), to achieve partial or complete bond disruption. Not only are such conditions generally harmful to any bound moiety (e.g. a protein or nucleic acid molecule), but these also result in denaturing of the biotin and/or streptavidin molecules. A denatured streptavidin molecule cannot be re-used. Moreover, since proteins can only be recovered under denaturing conditions these are inappropriate for the purification of delicate proteins. The use of denaturing conditions to reverse the biotin-streptavidin linkage is therefore undesirable, especially in bioseparations.
U.S. Pat. No. 5,387,505 describes a method for the separation of a complex comprising a biotinylated target nucleic acid and avidin-coated polymeric particles. This method involves heating of the complex to temperatures of at least 65° C., e.g. 85-100° C., in the presence of a salt wash solution comprising inter alia sodium chloride, SDS and EDTA. These harsh conditions similarly result in denaturation of the biotin and avidin molecules such that these cannot be re-used.
Others have reported various approaches to disrupt the biotin-streptavidin complex under more mild conditions, such as introducing light sensitive biotin phosphoramidites (Olejnik et al., Nucleic Acids Res. 24:361-366, 1996) or the use of polymer conjugates together with streptavidin mutants that yields temperature or pH dependent release. For example, Ding et al. (Bioconjugate Chem. 10:395-400, 1999) have conjugated a temperature-sensitive polymer, poly(N-isopropylacrylamide) (NIPAAm), to a genetically engineered streptavidin (SAv) to produce a conjugate capable of binding biotin at room temperature or lower and releasing bound biotin at 37° C. This conjugate can repeatedly bind and release biotin as the temperature is cycled through the lower critical solution temperature (LCST) of the polymer. More recently, Bulmus et al. (Bioconjugate Chem. 11:78-83, 2000) conjugated a pH-sensitive polymer (a copolymer of NIPPAm and acrylic acid) to the same specific site on the genetically engineered SAv molecule. Lowering the pH was found to cause the polymer to collapse leading to blockage of biotin binding, whereas raising the pH caused the polymer to fully hydrate thereby permitting biotin to bind.
However, none of the methods so far proposed for the reversible binding between biotin and streptavidin readily lends itself to use in DNA sequencing methods, for example using capillary or slab gel DNA sequencing instrumentation. Consequently, there is a continuing need in the art for alternative methods for reversibly and reliably disrupting the binding of biotin and streptavidin (or avidin), in particular such methods which use relatively mild conditions and so facilitate re-use of a streptavidin-coated support, e.g. in automated systems for DNA sequencing.